PLC-beta isozymes are stimulated by direct interaction with activated heterotrimeric G proteins as well as Rac isozymes of the Rho family of GTPases. Active PLC-beta isozymes catalyze the hydrolysis of phosphoinositides leading to the release of calcium stores and stimulation of protein kinase C, which are required for a variety of normal cellular processes. Conversely, abnormal signaling through PLC-beta leads to a variety of diseases including acute myeloid leukemia, cardiac hypertrophy and hypertension, and ataxia. The focus of this proposal is to determine mechanisms of regulation of phospholipase C-beta (PLC-beta) by Rho GTPases and heterotrimeric G proteins. In the first aim, crystal structures of isolated PLC- beta2 as well as PLC-beta2 bound to Gbetagamma will be determined and compared with the existing structure of PLC-beta2 bound to Rac to define modes of PLC-beta activation at atomic resolution. In the second aim, these structural analyses will be combined with existing data to generate mutant proteins to manipulate PLC-beta activation in vitro and in vivo. Cumulatively, these proposed studies are designed to provide a coherent framework for understanding and manipulating PLC-beta activation by various modulators for the ultimate goal of treating attendant diseases.